Targeted DNA sequencing of cell-free tumor DNA (circulating tumor DNA, ctDNA) shed into a patient?s blood holds great promise for detection, diagnosis, and monitoring of cancer. Given that all tumor cells have the potential to shed ctDNA, targeted assays using modern next-generation sequencing methods and digital PCR can provide insight into the entirety of a patient?s tumor burden. ctDNA is therefore an attractive biomarker for diagnosis and monitoring of cancer patients via non-invasive peripheral blood draw. Such assays, however, require ultra-sensitive sequencing fidelity, flexible target multiplexing, and uniform target amplification for efficient, quantitative, and cost-effective testing. In addition, several challenges arise in the implementation of assays to identify and monitor tumor-specific mutations extracted from blood. First, the mutant DNA from cancer cells is rare compared to the background of normal DNA, requiring detection sensitivities below 0.1% for some clinical applications. Second, cell-free DNA extracted from plasma is limited in mass (<100 ng / 5 ml sample), and typically degraded to very short fragment lengths, requiring efficient capture and amplification of the targets of interest. Third, cancer diagnosis and monitoring may require surveillance of a multitude of tumor or patient-specific mutations, requiring multiplex amplification for efficient sample utilization. In collaboration with our academic partners at Boston University and the University of California San Francisco, we have developed a novel next-generation sequencing sample preparation platform that addresses these critical challenges - PIPSenSeq (Pre-templated Instant Partitions for Sensitive Sequencing). This approach takes advantage of molecular indexing for consensus-read sequencing in combination with single-molecule amplification in Poisson-distributed nanoscale partitions. Furthermore, PIPSenSeq provides a simple, rapid library preparation that does not require complex, expensive instrumentation or microfluidic consumables. In this proposal we will develop PIPSenSeq as a commercial-ready platform for cancer monitoring, and demonstrate clinical utility in a study of 60 - 70 head and neck squamous cell carcinoma patients. These patients will be monitored post-operatively for tumor recurrence using personalized PIPSenSeq panels on longitudinally collected ctDNA samples.